One-pot synthesis, spectroscopic characterizations, quantum chemical calculations, docking and cytotoxicity of 1-((dibenzylamino)methyl)pyrrolidine-2,5-dione

Article abstract of DOI:10.1016/j.molstruc.2019.127403

The investigations of stabilization energy, bond order, intra-molecular interactions and energy gap of pharmaceutical drugs are useful for understanding their behaviour. A new Mannich base molecule 1-((dibenzylamino)methyl)-pyrrolidine-2,5-dione (SBF) was

Full text of DOI:10.1016/j.molstruc.2019.127403

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One-pot synthesis, spectroscopic characterizations, quantum chemical
calculations, docking and cytotoxicity of 1-((dibenzylamino)methyl)pyrrolidine-2,5-
dione
Krishnan Sarojini Devi, Palaniappan Subramani, Shanmugasundaram Parthiban, Namadevan
Sundaraganesan
PII:
S0022-2860(19)31512-1
DOI:
https://doi.org/10.1016/j.molstruc.2019.127403
MOLSTR 127403
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
19 June 2019
11 November 2019
Please cite this article as: Krishnan Sarojini Devi, Palaniappan Subramani, Shanmugasundaram
Parthiban, Namadevan Sundaraganesan, One-pot synthesis, spectroscopic characterizations,
quantum chemical calculations, docking and cytotoxicity of 1-((dibenzylamino)methyl)pyrrolidine-
2,5-dione, Journal of Molecular Structure (2019), https://doi.org/10.1016/j.molstruc.2019.127403
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Graphical Abstract
One-pot synthesis, spectroscopic characterizations, quantum
chemical calculations, docking and cytotoxicity of
1-((dibenzylamino)methyl)pyrrolidine-2,5-dione
Krishnan Sarojini Devi a, Palaniappan Subramani b, *, Shanmugasundaram Parthiban a,
Namadevan Sundaraganesan c
a Department of Chemistry, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
b Chemistry Section, Faculty of Engineering and Technology, Annamalai University,
Annamalainagar 608 002, Tamil Nadu, India
c Physics Section, Faculty of Engineering and Technology, Annamalai University,
Annamalainagar 608 002, Tamil Nadu, India

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One-pot synthesis, spectroscopic characterizations, quantum
chemical calculations, docking and cytotoxicity of
1-((dibenzylamino)methyl)pyrrolidine-2,5-dione
Krishnan Sarojini Devi a, Palaniappan Subramani b, *, Shanmugasundaram Parthiban a,
Namadevan Sundaraganesan c
a Department of Chemistry, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
b Chemistry Section, Faculty of Engineering and Technology, Annamalai University,
Annamalainagar 608 002, Tamil Nadu, India
c Physics Section, Faculty of Engineering and Technology, Annamalai University,
Annamalainagar 608 002, Tamil Nadu, India
*Corresponding author
Dr. P. Subramani
Professor
Chemistry Section
Faculty of Engineering and Technology
Annamalai University
Annamalainagar 608 002.
Tel.: +91 9443536358
E-mail: pschem2018@gmail.com

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2
One-pot synthesis, spectroscopic characterizations, quantum
chemical calculations, docking and cytotoxicity of
1-((dibenzylamino)methyl)pyrrolidine-2,5-dione
Krishnan Sarojini Devi a, Palaniappan Subramani b, *, Shanmugasundaram Parthiban a,
Namadevan Sundaraganesan c
a Department of Chemistry, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
b Chemistry Section, Faculty of Engineering and Technology, Annamalai University,
Annamalainagar 608 002, Tamil Nadu, India
c Physics Section, Faculty of Engineering and Technology, Annamalai University,
Annamalainagar 608 002, Tamil Nadu, India
ABSTRACT
The investigations of stabilization energy, bond order, intra-molecular interactions and
energy gap of pharmaceutical drugs are useful for understanding their behaviour. A new
Mannich base molecule 1-((dibenzylamino)methyl)-pyrrolidine-2,5-dione (SBF) was
synthesized through sequential Mannich reactions of an imide with an aldehyde and the
intermediate obtained are reacted with secondary amine to get the desired product.
The entire molecular geometry of the SBF molecule was confirmed by single XRD
studies. The crystal structure of SBF molecule was found symmetrical and planar with
anti-periplanar orientation of C=O group and nitrogen atom. The theoretical computations
were performed using B3LYP/6-31G(d, p) level of basis set. The chemical environment of
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1
carbon and protons were shown with C and H NMR spectral analysis, respectively.
The molecular weight of the title molecule was determined by mass spectrum. The thermal
stability of the SBF molecule up to 270 C was exposed by thermogravimetric (TG) and
differential scanning analysis (DSC). The natural bonding orbital (NBO) analysis data
revealed the highest stabilization value of E(2) for transfer of electron density from
succinimide to the antibonding molecular orbital of the dibenzyl ring were found to be
51.51 and 50.31 kJ/mol. The energy of HOMO-LUMO, PDOS, OPDOS, RDG of the title
molecule have also been carried out. The protein-ligand relationship was well revealed by
molecular docking studies. The cytotoxic activity of the SBF molecule was evaluated by
MTT assay on cell line A549.
Keywords: Mannich reaction; Intermolecular interactions; Differential scanning analysis;
Energy gap; Molecular docking; Cytotoxic activity

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1. Introduction
The Mannich reaction is in terms of nucleophilic addition reaction, based on the
condensation reaction of a compound incorporating hydrogen(s) which are active along
with a primary or secondary amine and any one of the aldehyde [1]. The N-Mannich
reaction basically happens in three component condensation involving the primary or
secondary amine. The reaction forms the carbon bridge through which the condensation
reaction occurs with an electron-deficient nitrogen functional group (amide, sulfonamide,
imide, etc.). The carbon bridge is the mostly formed by methylene derived using generally
formaldehyde; the reaction final product represents as an N-Mannich base. This amino-
methylation reaction occurs at vigorous condition and intense protic solvent setting to
get the final product. The synthetic method of Mannich base compounds was recognized
as a versatile origin because it opens the door to derive biologically significant
pharmaceutical products [2–5]. They discover many uses such as antimalarial, antitumour,
antimicrobial, antitubercular, antiinflammatory, anticonvulsant agents [6], vasorelaxing [7],
and analgesic drugs [8]. At the moment, Mannich base reactions in chemistry, suggest
many opportunities molecular structure to create biologically active and it is dependent on
the prospect and their requirements. The Mannich base molecular system provides an
interaction of transition metal ions, which are the most vital region in the coordination
chemistry. Several Mannich bases and their metal complex geometrical structures have
their own resourcefulness of biological significance and they play an essential role in
industrial applications.
The significations of N-Mannich bases like prodrug forms have formerly investigated
by Koch et al. [9]. For design, the N-Mannich base reactions of succinimide with aromatic
amines were recognized as prodrug for medicinal value. Their molecules contain an imide
ring and the general structure –CO–N(R)–CO–, being hydrophobic and neutral and they

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can therefore diffuse through biological membranes. Succinimide derivatives are
important in biology due to their pharmacological properties. Many Mannich bases are
found with remarkable biological activity. 1-[Anilino(phenyl)methyl]pyrrolidine-2,5-dione
is a Mannich base and its transition metal complexes have an expensive antimicrobial
activity. For illustration, 1-[(pyridin-2-ylamino)methyl]pyrrolidine-2,5-dione under
investigation has its individual biological merits chiefly on screening antioxidant
activity. The Mannich bases of 2-propoxybenzylideneisonicotinohydrazide derivatives
shows cytotoxicity for the A-549 human lung cancer cell line by Bala et al. [10]. The pyrrolidine
ring structure is most valuable for bioactive natural products since it increase activity of
drugs. Recent studies reveal that these type of molecules are used as organocatalysts with
broad range of metal-mediated enantioselective protocols [11]. The cancer activity of
N-Mannich base molecule on human cancer cell lines A-549 based on SRB assay method
were reported by Piplani et al. [12]. The cytotoxicity of Mannich base were reported by
Muruganandam and Maheswari [13] and Dimmock et al. [14]. In recent years, the treatment
of cancer has altered from classical to targeted and better tolerated chemotherapy [15].
Antiproliferative activity of 2-(n-alkylamino)-1,4-naphthoquinone derivatives was evaluated
by Patil et al. [16] and Neves et al. [17]. The breast and colon activity of cell line MDA-MB-231
and HCT-116 was available by given literature [18]. The cytotoxicity of reduced form of
n-alkylamino analogous structure of vitamin K3 are reported by Chadar et al. [19].
Moreover, the newly synthesized Schiff base molecules are effective corrosion inhibitors by
Saha and Banerjee [20]. The American Cancer Society investigated that 228,150 new cases
of lung cancer in U.S is identified and 142,670 death leads to lung cancer happen in
2019. We have completed to report probably for the first time, the results based on
quantum chemical calculations, HOMO-LUMO, electronic structure, hybridization and
intermolecular interactions on SBF molecule and also the first time reported the crystal
structures of SBF molecule obtained from the addition of three reactants such as imide,

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aldehyde and secondary amine in acetonitrile medium. The synthesized SBF molecule was
also characterized using NMR, HRMS, TG-DSC, etc., spectral techniques. The molecular
characteristics such as NBO analysis, energy gap, ionization potential, electron affinity,
electronegativity, absolute hardness, chemical potential, softness, nucleophilicity, etc. of
SBF molecule are described via quantum chemical method. The cytotoxic activity of the
SBF molecule is resolved by MTT assay on cell line A549.
2. Experimental and computational details
2.1. Synthesis of 1-((dibenzylamino)methyl)pyrrolidine-2,5-dione (SBF)
The raw chemicals employed in the synthesis of SBF were purchased from Alpha and
Aldrich Chemical Company with spectroscopic grade of purification. Hence there is no
any need for further purification on reactants such as succinimide, formaldehyde and
dibenzylamine along with solvent used all the way through various stages of the synthesis.
1-[(Dibenzylamino)methyl]pyrrolidine-2,5-dione (SBF) was synthesized by Mannich
condensation reaction between succinimide, dibenzylamine and formaldehyde in 1:1:1
molar ratio (Scheme 1). Succinimide (5.00 g, 0.05 M) and formaldehyde (4.5 mL, 0.05 M)
mixed with the least amount of acetonitrile were stirred until the content becomes
homogenous. To this mixture dibenzylamine solution (3.8 mL, 0.05 M) was poured slowly
in drops while the solution is stirred. Acetic acid (0.01 M) was added and the reaction
mixture was stirred with reflux condition for 10 h. The completeness of the reaction was
proved by TLC and cooled at room temperature. The reaction solution was poured into
ice, the solid mass obtained was rinsed using distilled water and cold ethanol, desiccated
and the final residue was recrystallized from acetonitrile and chloroform (4:6) to yield a
colourless crystalline cubic sized SBF (yield: 95%). The SBF molecule is a colourless
crystalline solid and has a melting point of 121 C. The SBF molecule shows a pH of
6.98 in ethanol medium.

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2.2. Spectroscopy measurements
Melting point was recorded using a melting point. The X-ray intensity data were
collected at temperature of 296 K on a Bruker D8 Venture SC-XRD equipped with X-ray
generator operating at 45 kV and 10 mA for Mo- and CuK radiation of wavelength
1.54178 Å. A complete data set was processed using SAINT PLUS. The structure
(SBF molecule) was solved by direct methods and refined by full-matrix least squares
method on F2 using SHELXS and SHELXL programs. The geometrical calculations were
carried out using the program PLATON. The powder XRD data were collected in a Philips
1
instrument model PW 1830 using CuK radiation ( = 1.5406 Å). H NMR spectra of a
molecule were recorded on Bruker 500 MHz Avance NMR spectrometer using TMS as an
internal standard and its processing resonance frequency with the applied radio-wave
frequency at 0 ppm in -scale (the chemical shift value). Furthermore, the sampling of
the compound was done using highly polar protic solvent, dimethylsulfoxide (DMSO).
Proton decoupled 13C NMR spectra were recorded on NMR Bruker Avance 500 MHz
spectrometer with an operating frequency of about 125 MHz, using the deuterated DMSO.
TMS was used as an internal standard. The HRMS analysis was done using high
resolution Q-TofMass, mass spectrometer as detector (Perkin Elmer), TurboMassver 5.4.2
software for data analysis. The GC parameters were found as split 10:1 injection; column:
Elite-5MS (30 m  0.25 mm I.D, 250 m df); injection port temperature: 250 C; carrier
gas: helium; flow rate: 1 mL/min; oven temperature: initially 60 C for 2 min, ramped to
300 C at 10 C/min, 300 C for 6 min. The MS conditions found as follows: transfer line
heater: 240 C; ion source temperature: 240 C; electron impact ionization (EI) mode;
ionization energy: 70 eV. Analysis was performed in scan mode, from 600-10 amu at a
speed of 1.5 scans/s. Solvent delay was set as 2.0 min as observed. The obtained mass
spectra were compared with commonly used EI-MS spectra libraries. TG/DSC study was
engaged with the thermo gravimetric instrument TGA7 (Perkin Elmer) Q500 Hi-Res TGA

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from TA instruments), DSC7 (Perkin Elmer) Q200 Hi-Res TGA from TA instruments.
UV-Vis spectrum was recorded in DMSO solvent by employing the Lambda-35 Perkin
Elmer spectrophotometer as a part of the spectral locale of 1100–190 nm.
2.3. Theoretical calculations
Crystal packing diagrams were obtained using Mercury software [21]. Crystal
Explorer 3.1 program was used to perform the Hirshfeld surfaces theoretical analysis [22].
Theoretical predictions were evaluated by Khon-Sham’s DFT method, subjected to the
gradient-corrected hybrid density functional B3LYP method [23,24]. For the structure, the
6-31G (d, p) basis set [25] was used for a full geometry optimization using the published
function [26], which is implemented by Gaussian 09 package [27]. The structure of the
1
13
title molecule was visualized by GaussView 5.0.9 [28]. The H and C NMR chemical
shifts of the SBF molecule have been obtained from the Gauge-Independent Atomic
Orbital (GIAO) [29,30] approached by subtracting the shielding constants of SBF molecule.
The UV spectra were calculated using the TD-DFT [31,32] functional with 6-31G(d, p)
basis set in DMSO solvent using PCM model. The DOS data were calculated using
GaussSum 3.0 software [33]. The Multiwfn [34] and VMD program [35] were used to describe
the intramolecular interaction within a molecule. The NBO values were obtained with NBO 3.1
program [36], as implemented in the Gaussian 09 package using the same theory level.
2.4. Docking studies
The molecular docking study was investigated by using lung cancer and diabetic
proteins to find the active binding sites and interaction properties of amino acids with
synthesized molecules. The 3D crystal structure of protein complex (PDB format) was
downloaded from Protein Data Bank (www.rcsb.com). The protein complex and ligand
were organized after pre-processing by protein preparation wizard and ligand preparation
wizard [37] in Maestro 9.3.5 version of Schrodinger software.

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2.5. In vitro anticancer studies
A549 (Lung Cancer) cells were initially procured from National Centre for Cell
Sciences (NCCS), Pune, India and maintained by Dulbecco’s modified Eagles medium,
DMEM (Sigma Aldrich, USA).
The cell line was cultured in 25 cm2 tissue culture flask with DMEM supplemented
with 10% FBS, L-glutamine, sodium bicarbonate (Merck, Germany) and antibiotic
solution containing: Penicillin (100 U/mL), Streptomycin (100 µg/mL), and
Amphotericin B (2.5 µg/mL). Cultured cell lines were kept at 37 C in a humidified 5%
CO2 incubator (NBS Eppendorf, Germany). Anticancer studies were carried out by
Biogenix Research Centre, Poojapura, Trivandrum.
The viability of cells were evaluated by direct observation of cells by inverted
phase contrast microscope and followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay method.
2.5.1. Cell treatment procedure
Two days old confluent monolayer of cells was trypsinized and the cells were suspended
in 10% growth medium. 100 L cell suspension (5104 cells/well) was seeded in 96 well
tissue culture plates and incubated at 37 C in a humidified 5% CO2 incubator.
2.5.2. Preparation of compound stock
1 mg of sample was weighed and dissolved in 1 mL DMEM using a cyclomixer.
The sample solution was filtered through 0.22 m Millipore syringe filter to ensure the sterility.
2.5.3. Anticancer evaluation
After 24 h the growth medium was removed, each freshly prepared compounds in 5%
DMEM were serially diluted five times by two fold dilution (100, 50, 25, 12.5, 6.25 g in
500 L of 5% DMEM) and each concentration of 100 L were added in triplicates to the
respective wells and incubated at 37 C in a humidified 5% CO2 incubator. Non-treated
control cells were also maintained.

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2.5.4. Anticancer assay by direct microscopic observation
Entire plate was observed after 24 h of treatment in an inverted phase contrast tissue
culture microscope (Olympus CKX41 with Optika Pro5 CCD camera) and microscopic
observation were recorded as images. Any detectable changes in the morphology of the
cells, such as rounding or shrinking of cells, granulation and vacuolization in the
cytoplasm of the cells were considered as indicators of cytotoxicity.
2.5.5. Anticancer assay by MTT method
Fifteen mg of MTT (Sigma, M-5655) was reconstituted in 3 mL PBS until completely
dissolved and sterilized by filter sterilization. After 24 h of incubation period, the sample
content in wells were removed and 30 L of reconstituted MTT solution was added to all
test and cell control wells, the plate was gently shaken, then incubated at 37 C in a
humidified 5% CO2 incubator for 4 h. After the incubation period, the supernatant was
removed and 100 L of MTT solubilization solution (dimethylsulfoxide (DMSO), Sigma
Aldrich, USA) was added and the wells were mixed gently by pipetting up and down in
order to solubilize the formazan crystals. The absorbance values were measured by using
microplate reader at a wavelength of 540 nm [38]. The percentage of growth inhibition
was calculated using the formula:
Mean OD samples
% of viability =
100
Mean OD of control group
2.6. Statistical analysis
The IC50 values were measured from plots of percentage inhibition versus log
inhibitor concentration and were calculated by nonlinear regression examination from the
mean inhibitory values. In an anticancer research the nonlinear regression graph was
plotted between percentage of cell inhibition and Log10 concentration. IC50 was
determined using the Graph Pad Prism program.

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3. Results and discussion
3.1. Single crystal diffraction
The SBF molecule was found to have an empirical formula C19H20N2O2, colourless,
monoclinic space group P21/c with a meagre second harmonic activity. The molecular
geometrical structure with atom numbering system of SBF molecule at 40% thermal
dislocation ellipsoid and molecular optimized structure is shown in Fig. 1. The molecular
packing diagram of the SBF molecule is shown in Fig. 2. The molecular and packing
diagrams were produced by Mercury software [21]. The SBF molecule consists of
electronegativity nitrogen atoms bonded with the carbon (C30) atom of dibenzyl moiety.
The succinimide ring (C=O) consists of carbonyl group acts as an electron acceptor
with other termination. This SBF molecule contains twenty C–H bonds, seventeen C–C
bonds, two C=O bonds and six C–N bonds. The unit cell parameters are calculated such as
a = 14.1336(3) Å, b = 5.54310(10) Å, c = 21.4306 Å, α = 90, β = 103.529, γ = 90.
The unit cell volume is found to be 1632.37 Å3 as shown in Table 1. There are two
independent structure (succcinimide and dibenzyl ring) in the asymmetric parts of the
crystallized SBF molecule. The molecular structures of the asymmetric units of the SBF
molecule are stabilized through intra-molecular hydrogen bonding COH interactions.
The hydrogen bonding interaction involves carbonyl group of succinimide (C–O42) and
hydrogen atom of benzyl group, with a distance of 3.265 Å as shown in Table 2. The
crystal packing structure is subjected by CH– (edge-to-face -stacking) and offset –
stacking interactions, which usually are the major forces leading the crystal structure of
aromatic systems [39]. These two classes of interactions are recognized by both benzyl
rings and the succinimide group. Each succinimide group interacts through offset –
stacking with another succinimide group of a neighbouring molecule, with a distance of
3.015 Å between the planes containing each heterocycle. On the other hand, one of the

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benzyl groups acting as the H donor makes the CH edge-to-face -stacking interaction
with a neighbouring benzyl ring of a closest molecule, calculating 2.561 Å between the
centroid of the charge donor ring and the hydrogen atom involved in the interaction.
On the contrary, the other benzyl group within the similar molecular unit acts as double H
acceptor and interacts through CH edge-to-face -stacking simultaneously with a
succinimide aromatic unit (3.111 Å between the centroid of the ring and the hydrogen
atom) and a benzyl group (2.854 Å between the centroid of the ring and the hydrogen
atom) of two adjacent molecules, resulting in a double T-shape -stacking interaction.
This double T-shape interaction related to the benzyl ring with the largely deviation
from the predicted molecular structure, which was investigated under the isolated
molecule estimation in which the crystal packing effects are not taken into account.
The SBF molecule was crystallized with the prominence to recognize the internal
molecular geometry and van der Waals interaction in the crystal structure of molecule.
The van der Waals interactions among with the molecules create further stability of the
crystal structures. The entire H atoms were positioned geometrically and constrained to
ride on their parent atom with C–H = 0.93–0.97 Å.
3.2. Powder X-ray diffraction
The powder X-ray diffraction (PXRD) of SBF molecule shows the sharp peak and the
simulated peak available from Mercury software [21] (Figs. 3a and 3b). The different
planes of reflections in powder X-ray diffraction pattern were also indexed with the help
of the same software. From the graph, it is clear that the both observed and theoretical
patterns are good concurrence with each other. These results will be helpful to identify the
crystalline phase with a simple PXRD pattern relatively than repeating the single crystal
X-ray diffraction method. The sharp peak clearly reveals the good crystallinity nature of
the SBF molecule.

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3.3. Hirshfeld surface analysis
To recognize the intermolecular interactions such as H–H, O–H, C–H, O–O, N–H,
N–O, O–C contacts, the Hirshfeld surface of the SBF molecule was obtained by well known
crystal explorer method 3.1 [22], which used the crystallographic information file (CIF) as
an input. The 2D fingerprint plot consist of two dimensional (de, di) contacts, where de
represents the distance of the adjacent nucleus exterior to the surface and di indicates the
distance from a region on the surface to the nearby nucleus inside the surface on Hirshfeld
surface. The dnorm mapped on the Hirshfeld surfaces were generated with colour scale in
between –0.080 a.u (blue) to 1.593 a.u (red) respectively. Similarly, for di, de, shape index
surfaces and curvedness surface were generated with colour scale 1.000–2.468, 1.000–2.412,
–1.000–1.000, –4.000–0.4000 a.u respectively as shown in Fig. 4. The Hirshfeld three-
dimensional dnorm surface of the SBF molecule (Fig. 4c) reveals strong interactions (light
red to white) [40]. The red regions labelled in the dnorm map are attributed to OH/HO
contacts. The pale blue to white spot indicates CH/HC contacts associated with strong
CHC and the light spots related to HH interactions. The close assessment with shape
index and curvedness was also analyzed. The deep red circle indicated by concave regions
on the shape index surface (Fig. 4d) explains the hydrogen-bonding contacts, which
corroborates the occurrence of an intramolecular hydrogen bond between carbonyl groups
and benzyl ring. The blue triangles implied by convex regions on the shape index surfaces
have the characteristics of π–π stacking of aromatic molecules [41]. The existence of π–π
interactions is also evident from the relatively huge and green flat regions enclosed by
blue circles, on the consequent curvedness surfaces (Fig. 4e) [42].
The existence of relatively flat regions on the curvedness surface above the picrate
moiety also displays the scope of planar stacking in SBF molecule. The de and di distance
scales are displayed in the range of 0.6–2.4 Å in the 2D fingerprint plots [43]. The HH

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interaction reflected in the 2D fingerprint plots is displayed fingerprint plot which has the
highest contribution of 61.7% and carbonyl groups and phenyl ring enhances the CH and
OH/HO interactions (Fig. 5). The contributions are found lesser than the HH
interaction 22.2 and 13.8% to the total Hirshfeld surfaces shown as a broad blue region.
Fig. 5d represents the weakest contributions of OO, OC, NO, NH intramolecular
bonding interactions. The presence of lone pairlone pair (OO (1.0%)), lone pair
(OC (0.7%), NO (0.4%)) and lone pair (NH (0.2%) and CC, CN, NN
(0.0%)) interactions are also observed. Here the value of de and di is less than the van der
Waals radius of hydrogen atom (2.4 Å) which indicates the presence of HH non-covalent
interaction in this compound.
3.4. NMR spectral analysis
Quantum chemical calculations through density functional theory (DFT) are precise
enough in the prediction of NMR spectra, which explores the association between the
molecular structure with their chemical shifts [44,45]. The chemical shifts of the SBF
molecule are optimized by using DFT-B3LYP method and GIAO approach with 6-31G(d, p)
as basis set [46,47]. The solvent induced effects are studied by using the IEFPCM model
with CDCl3 solvent [48]. The chemical shifts of SBF molecule; observed and simulated
are given in Table 3, with their assignments. The proton and carbon NMR spectra of the
SBF molecule in CDCl3 solvent are shown in Fig. 6.
3.4.1. 1H NMR
The singlet peak shows the presence of equivalent proton of N–CH2–N (H31 and H32)
appeared as having the highest chemical shift value (4.52 ppm), due to deshielding effect
by electronegative nitrogen atom (N29, N31) and electron deficient group of carbonyl atom
(C=O). Similar shift in the electron donating and electron attracting group of SBF

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molecule has been observed. The singlet peak for equivalent proton of Ar–CH2–N (H2, H3,
H5 and H6) is appeared at highest chemical shift value (3.74 ppm), on account of
deshielding effect of electron-withdrawing group like nitrogen atom (N29). The singlet
signal is appeared at deshielded region for electron deficient group of carbonyl atom
(C=O) at 2.52 ppm corresponding to the equivalent methylene protons of the succinimide
ring. From the literature survey, the 1H NMR spectrum values for the aromatic protons are
1
generally observed at ~7.0–12.0 ppm. The H NMR chemical shift values of aromatic
proton in SBF molecule at 7.39–7.21 ppm are in good agreement with the theoretical
values in gas phase (7.06–7.69 ppm) and in solvent phase (7.18–7.87 ppm). In SBF
1
molecule, the H NMR spectrum shows three multiplet peaks in the region 7.39–7.37,
7.32–7.29, 7.26–7.29 ppm for aromatic protons.
3.4.2. 13C NMR
In 13C NMR spectrum, the carbon atom of carbonyl group in succinimide ring
appeared at the most shifted δ values area is 177.9 (experimental) and 183.01, 181.10 ppm
in gas phase and 185.03, 184.40 ppm in solvent phase. The carbon atom of functional
group (C=O) found at the most deshielded area compared to other carbon atoms is due to
the presence of most electro-negativity oxygen atom that decrease the electron density
distributed of carbon atom. The aromatic carbon atoms in phenyl rings are established at
128.2–128.6 ppm and the theoretical δ values of aromatic carbon atoms are in the range of
132.36–135.92 ppm in gas phase and 131.37–134.83 ppm in solvent phase. The peak
appeared at 139.2 ppm for C7 and C18 are assigned to ipso carbon of phenyl group. In SBF
molecule, the NMR spectrum shows three aliphatic regions, the peak appeared at
58.0 ppm (C30) seem to have higher chemical shift value as predicted with the theoretical
value at 68.79 ppm in the gas phase and 64.12 ppm in solvent (CDCl3) phase. The
presence of higher electronegative nitrogen atom on both sides gives the electron

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distribution around the bond to polarize through the adjacent carbon atom; the electron
density was found decreased shifted towards downfield region. The other peak is appeared at
higher wavenumber due to the presence of electron withdrawing group of nitrogen atom (N29)
which is attached to carbon atoms C1 and C4. Thus the result of NMR signal was observed
in the downfield at 56.6 ppm and the calculated values at 60.52 and 63.79 ppm in gas
phase and in solvent phase at 55.06 and 58.09 ppm. From Table 3, it should be observed
that the chemical shift value of the C34 and C35 atoms at 28.2 ppm to the electron
deficient group of carbonyl atom (C=O) atoms in the non-aromatic part of the SBF
molecule is observed in 35.21, 33.50 ppm in gas phase and 31.34, 29.75 ppm in solvent phase.
The CDCl3 (solvent) peak was appeared at 40.24–39.48 ppm as shown in Fig. 6b.
3.5. Mass spectrum
The mass spectrum of SBF analysis was carried with HRMS method. In common, the
calculated mass was presented at 308.1525 (100.0%) high predominant, 309.1558 (20.5%)
and 310.1592 (2.0%) less predominant. Similarly, the experiment mass with less
predominant is also referred to as a base peak, at 309.1573 (100%) coincided with related
molecular weight (309.1558). The mass spectrum and fragmentation pattern of SBF
molecule are shown in Fig. 7. This peak is represented to as the most stability molecular
ion peak [C19H21N2O2] of SBF molecule.
3.6. TG/DSC analysis
The thermal stability of the SBF molecule was recognized by TGA and DSC
thermogram depicted in Fig. 8. The thermogravimetric analysis of the SBF molecule in air
by the heating rate of 20 C/min in nitrogen atmosphere, the temperature range was found
between 0 and 600 C. From the TGA curve, it is clearly noted that until reaching the
melting point (121 C), there is no mass loss and it indicates as the unreality of crystallization

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solvent or contamination of SBF molecule up to onset of melting point. The smooth curve
response of TGA plot behind its melting point corresponds to the thermal decomposition
of SBF with the creation of reaction products [49]. From the TG curve, it is inferred that
the sample of the SBF molecule undergoes decomposition in three stages. This nature of
weight loss exposes there is no volatilization with any fragmentation. The SBF molecule is
stable up to 270 C and no phase transition occurs till the melting point of the crystal.
There is a major weight loss 82.97% is observed in the temperature range from 270.6 to
294.4 C. The mass loss is due to the liberation of volatile benzyl ring products. The weight
loss takes place from 294.4 to 415.7 C on the account of the unpredictable product like
methylene radical and residue left about 14.37% found towards the end. The second broad
exothermic peak appears at 294.4 C and behind this step the weight loss in TGA occurs
progressively. The decomposition temperature has 294.4 C in TGA graph, behind its
melting point, corresponds to the thermal stability of the SBF molecule.
The mass loss below 294.4 C represents the SBF molecule as devoid of physically
adsorbed water and water of crystallization. The residual carbon mass at 575.4 C at the
end of the composition reaction is 0.22%. From the graph, it is the sharp and broad
exothermic peaks appear at 123.3 and 282.9 C. The exothermic peak related to the
activity of the sample melting point liberating the facilitating the process of melting (123.3 C).
The second broad exothermic peak was shown at 282.9 C. The most curve value and
height of the curve will be represented by mass of the material and perhaps by
encapsulation procedure, since the bad thermal conduct forms lower curve height with
broader peak [50,51]. It is definite by the purity of the material and to describe the peak
available by a DSC curve with its shape. The higher concentration of the contamination in
a given substance lower the melting point and broader the range. In SBF molecule, the
maximum melting point and the sharpness in DSC melting curve expose that SBF has
higher crystallinity and purity.

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3.7. UV-visible spectral analysis
Optical transparency in the visible and near infrared region (400–1100 nm) and low
transparency cut off wavelength (<400 nm) are the essential requirements for an organic
NLO material. The second order NLO properties like frequency doubling of the incident
laser beam is observed in this region. Any electronic absorption in this region by an
organic molecule may mask the nonlinear optical properties. The electronic absorption
spectrum of SBF in chloroform in the UV-Vis-NIR region is recorded to determine the
optical transparency window and the transparency cut-off wavelength and to study the
effect of electronic delocalization in the molecule. Experimental and simulated absorption
spectra of the SBF molecule are shown in Figs. 9 and 10.
Theoretical electronic absorption wavelength as well as oscillator strengths of title
molecule were calculated by TD-DFT B3LYP/6-31G(d, p) level of theory for both gas
phase and solvent (chloroform) phase. The experimental absorption wavelengths (λ),
theoretical absorption wavelengths (λ), energy gap, oscillator strengths (f), excitation
energies (E) and major contributions to electronic transition are given in Table 4. From the
theoretical UV-Vis spectra, we have observed three bands at 353, 307, 304 nm in gas
phase and 353, 310, 306 nm in CHCl3 and the corresponding experimental peak
observed at 386 nm. The absorbed peak (λmax) is obtained at 353 nm with an oscillator
strength f = 0.0021, the energy gap value 4.7780 eV and HOMO-LUMO with 100%
contribution is responsible for electronic transition due to the assigned of nπ* transition.
The ES1 represents the transition between HOMO-82 and LUMO-83. It implies an
electron density transfer from the benzyl ring to electron withdrawing atoms like oxygen
and nitrogen (n–π*). The electronic hyperpolarizability and charge transfer involving in
the molecule leads to better light harvesting effectiveness in the SBF molecule.

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3.8. Frontier molecular orbital analysis
The energy gap is a decisive parameter to determine the molecular electrical transport
characters [52]. The conjugated molecules are demonstrated by the highest occupied
molecular orbital and the lowest unoccupied molecular orbital (HOMO–LUMO)
separation, which is an essential part of intra-molecular charge transfer (ICT) from the
end-capping electron contributors groups to the efficient electron receptor groups via
-conjugated way [53]. The energy gap between the HOMO and LUMO also explains the
chemical reactivity and the stability of the molecule. Therefore, the electron density is
transfer more from the dibenzyl moiety of the -conjugated structure act as an electron
donor side to its electron-withdrawing component. The title compound has 82 occupied
molecular orbitals and the frontier orbital energy gap of SBF molecule is determined by
TD-DFT method using 6-31G(d, p) basis set. The energy gap of the SBF molecule can be
calculated by ploting h against (h)2. The energy gap is observed at 2.65 eV and
compared with simulated data 3.51 eV (gas phase) and 3.51 eV (solvent phase) (Table 5
and Fig. 11). However, in present study the energy of HOMO is –5.94 eV (gas phase) and
–5.35 eV (solvent phase), whereas the energy of LUMO is –0.58 eV (gas phase) and –0.83
(solvent phase) and their energy gap is 5.36 eV (gas phase) and 5.35 eV (solvent phase) of
SBF molecule as summarized in Table 6. As can be seen from Fig. 12a, HOMO is located
over the whole skeleton of the succinimide molecule of carbonyl, nitrogen atoms and
methylene of dibenzyl ring except the phenyl ring and hydrogen atoms whereas LUMO is
fully spread over phenyl ring, oxygen and nitrogen atoms except succinimide and
hydrogen atoms. A molecule with a minimum energy gap will determine the good
chemical reactivity and since the molecule is said to be a softer whereas the molecule
with maximum energy gap is measured as a good thermal stability with high chemical
hardness [54–56]. This large energy gap implies that the structure of the title Mannich
base molecule is having good stability and chemical hardness [57].

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3.9. Total, partial and overlap population density of states
The density of states analysis has great importance in explanation of group donations
to electronic state energies. The most significant relevance of the DOS spectra is to
express molecular orbital (MO) compositions and their offerings to chemical bonding via
the OPDOS plots which are also available in the literature as crystal orbital overlap
population (COOP) maps. The OPDOS (or COOP) is related to DOS spectrum. The whole
density of states can be exhibited as the sum of density contributions from every group
during the partial density of states (PDOS) predictions. In the fact, the orbital energies
assigned in quantum chemical simulations are discrete, which are given as line graph
below PDOS graph, however, simulated drawing of density of states with a convinced
FWHM uses to interpret bonding situations. The overlap PDOS (OPDOS) displays
nonbonding (zero), the bonding (positive value) and antibonding (negative value)
interaction between the two orbitals, groups or atoms [58]. The DOS, PDOS and OPDOS
spectra are shown in Fig. 12. There are 82 orbitals in bonding and 362 antibonding MOS
having in the SBF molecule. Both the HOMO and the LUMO are the main molecular
orbital involving in the chemical reaction of a molecule. The energy gap with HOMO and
LUMO is an essential parameter to demonstrate the molecular electrical transport
character since it is used to find out the electron conductivity of a material. The PDOS
along with Mulliken population analysis was computed using the Gauss-sum program.
As a result of PDOS spectrum of SBF molecule can be fragmented into four groups which
are assigned as nitrogen, oxygen, carbon and hydrogen as shown in Fig. 12c. The highest
contribution comes from the nitrogen and oxygen and the least contributions from carbon
and hydrogen. Thus, the HOMO orbital (H, C, O and N) are represented as (11% + 23% +
2% + 65% = 100%) whereas LUMO (H, C, O and N) are represented as (7%+ 61% +32%
+1% =100%) as shown in Fig. 12c. However, the percentage contribution of atomic or

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molecular orbitals in the molecule is very hard to establish the bonding and antibonding
character. So we can use the OPDOS diagram and some of its orbitals of energy data
results of interaction among the selected groups which are obtained from figure easily to
identify the particular group of interactions as shown in Fig. 12b.
3.10. Reduced density gradient (RDG) analysis
The weak interactions in real space based on electron density and its derivatives have
investigated by Johnson and co-workers [59]. The weak interactions of a molecule can be
located on region as a low density and the RDG value becomes low. The plot of electron
RDG Vs ρ gives the strength of interaction. The sign λ2 is used to distinguish the bond
(λ2<0) interactions from non-bonding (λ2>0) interaction. The Multiwfn [34] and VMD
software [35] are helpful to calculate the RDG surface of SBF molecule. RDG value = 0.05
denotes that the molecule having both repulsive as well as attractive interaction as shown
in the Fig. 13. The value of RDG near zero means that the molecule on the equal
proportion of attraction and repulsion in the entire system. Single or several spikes are
present in the low electron density i.e., low-gradient region (Fig. 13a) which implies the
presence of weak non-covalent interactions. The low RDG value as well as weak
interaction can be placed by producing RDG isosurface enclosing the related regions in the
real molecular space (Fig. 13b). The gradient spikes isosurfaces are coloured with respect
to the value of sign(λ2)ρ as a good indicator strength. Higher negative value of (λ2)ρ shows
stronger attractive interaction and high positive implies repulsion. A value near zero
explains van der Waals weak interactions. As seen from Fig. 13, the red colour located on
the ring system is found to be steric effect, the green colour is due to the van der Waals (vdW)
interaction which gives low electron density in this position.

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3.11. NBO analysis
The hyperconjugative interaction, electron density moves from orbital having lone
pair of electron to vacant orbital and the valence space span was described by natural bond
orbital (NBO) analysis. The electron density delocalization with filled and unfilled NBO
orbitals can be interpreted by the second order perturbation theory, E(2) [60]. In our
current study, we have considered as the interaction of energies of a core and the lone pair
orbitals of SBF molecule to vacant orbitals, which are presented in Table 7. The highest
stabilization energy E(2) value influences the more intensive interaction among with
unfilled and filled orbital while the most donating ability of electrons, the extent of
elongation of the entire system might be greater. The probable interactions of the SBF
molecule are summarized in Table 7. The stabilization interactions between the lone pair
orbital and vacant orbitals are responsible for the characteristic feature of a pharmaceutical
compound and the biological activity of a molecule [61]. Also, the intramolecular charge
transfer of π-electron increases the polarity of the molecule and it could be responsible for
the NLO properties.
From Table 8, we analyzed the consequent bonds such as C1–N29, N29–C30, C30–N43,
C40–O42, N43–C33, C40–N43 and C30–O41. The  bond of C30–N43 is made of 0.5924(sp3.33
)
+ 0.8056(sp2.05). These values consider as a polarization coefficient (0.5924 and 0.8056) of
C30–N43 bond. The polarization coefficient of a C30–N43 is found higher with increase in
the electronegative character of this composition. The coefficient squares give the weight
as percentage (0.5924)2 = which represents to 23.09% occupied over on carbon atom. In a
similar method, unutilized oxygen has 76.78%. Therefore, the NBO result shows more
polarized towards the oxygen atom, since the electronegative properties of oxygen atom
are confirming in atomic charge analysis. The other relative bonds are conferred in Table 8
considering with hybridization and polarization coefficient.

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The significant hybrid orbital of the molecular system with low occupation numbers,
substantial p-character and the higher orbital energies includes π(C7–C8), n(2)O41,
π(C40–O41), π(C33–O42), n(1)N43 electron density energies –0.24426, –0.26964, –0.38865,
–0.38811, –0.27262, 99.96, 99.92, 99.88, 99.87, 99.77 and low occupation numbers
1.65686, 1.85540, 1.98744, 1.98866, 1.59256 and high occupation numbers are n(1)O42,
n(1)O41, σ(C40–O41), σ(C33–O42), σ(C7–C8), σ(C8–C10), σ(C12–C14) with p-character 42.51,
42.73, 57.16, 57.39, 63.32, 63.80, 63.93 and high occupation numbers 1.97677, 1.97581,
1.99451, 1.99465, 1.97277, 1.97745, 1.97908.
Thus, a very close to pure lone pair orbital participates in the electron donation
n(1)N43π*(C40–O41), n(1)N43π*(C33–O42), n(2)O42σ*(C33–N43), n(2)O41σ*(C40–
N43), π(C33–O42), n(2)O42σ*(C33–C35), n(2)O41σ*(C34–C40) and n(1)N29σ*(C30–N43)
interactions are found in the SBF molecule.
3.12. Bond order
Bond order is used to measure the number of electrons distributed in bonds between
pair of atoms present in a molecule. It shows number of chemical bonds between two
atoms within a molecule. The bond order provides the information about the stability of a
bond. It explains the degree of covalent bonded electrons pairs with in two atoms. From
molecular orbital theory, bond order is the quantity of charge positioned on a particular
bond, which is great importance for the calculation of dipole moment in a molecule.
Selected bond orders predicted at the B3LYP/6-31G(d, p) level by natural atomic orbital
theory and molecular orbital theory basis are tabulated to understand the bond order
relation as given in Table 9. Bond order defines which types of bonds present in the
molecule. For example the bond between carbon and nitrogen have three types of linkages
(CN, C=N, C–N). In SBF C–N type only exists. Bond order analysis reveals that N29–C30
bond order value is nearly equal to one (0.98).

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3.13. Molecular docking studies
The molecular structure of the novel Mannich base SBF molecule was performed by
density functional theory (DFT) predictions (B3LYP) with basis level 6-31G(d, p)
accessible in the Gaussian 09 package. In present study, we have designed Schrodinger
(Mastero 9.6) [37] obtained from Cambridge crystallographic data center, for probing
interacting protein space and ligand conformational space. Molecular docking is helpful to
explain the biomolecular interactions with the SBF molecule and the significant residue(s)
in the binding site of 2ITU and 2ITO. They are available with the probable bioactive
binding properties of the hit molecules with SBF as show in Fig. 14. The docking score of
the SBF molecule are –4.45 and–4.99 for 2ITU and 2ITO as shown in Table 10. The 3D
structure of SBF molecule shows hydrogen boding interaction with 2.07 and 2.15 Å
between carbonyl group of succinimide ring in SBF molecule with MET 793 amino acids
in the target protein 2ITU and 2ITO. The 2D structures of docking results of SBF
molecule clearly display interactions with amino acids MET 743 present in the active site
of 2ITU and 2ITO. This explains presence of hydrogen bonding interactions of carbonyl
group in SBF molecule with MET 793 and SER 98 in the active binding site of 2ITU and
2ITO. The docking results of SBF molecule conclude activity against the lung cancer as
compared from the cell inhibition on cell line A549.
3.14. In vitro cytotoxicity activities
The synthesized SBF molecule was evaluated for cytotoxic activity to find out the
effort of their improved partition coefficient. To resolve the cytoxicity, DMEM protocol
was employed and human cancer cell A549 was chosen for present study. The final results
have been determined IC50 values and percentage of control growth (Table 11 and Fig. 15).
Percentages of growth for choose cell lines is established to decrease with increase in
concentration ranging from 6.25 to 100 μg/mL. As a result, it concludes that cytotoxic
activity of SBF molecule shows activity for IC50 value of 176.27 μg/mL. So SBF molecule
has cytotoxic activity.

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3.14.1. Morphological study of A549 cell line
The SBF molecule has been treated with 6.25, 12.5, 25, 50 and 100 μg/mL of cell line
for 24 h and cell growth inhibition was analyzed using transmission electron microscope.
A549 cell line has round and regular with abundant organelles and normal double
membrane nuclei as shown in Fig. 16. After treatment of different doses with SBF
molecule, early stage apoptosis might be seen in Fig. 16. This effect slightly increases with
increasing concentration of SBF molecule. The shape of nuclear membrane was changed
from round to semi-circular, absence of sharp angle; nuclear chromatin was concentrated
and bundled on the inner border of Karyotheca. The endoplasmic reticulum has become
enlarged in interior wall. Finally, experimental results clearly show the cytotoxic activity
against the A549 cell line. It is interesting to observe from Fig. 16f the cytotoxicity of SBF
molecule has been increased as compared with control (Fig. 16a).
4. Conclusion
A new Mannich base SBF molecule was multi-component synthesized, spectral
characterized and its molecular geometrical structure was explained by X-ray single
crystal analysis. The crystal structure of SBF molecule exhibits monoclinic shape with
P21/c space groups. Intramolecular interactions in the crystal structure were studied in
detail using Hirshfeld surface analysis and 2D fingerprint plots, indicating that the most
frequent intermolecular interactions in the SBF molecule are reciprocal of HH,
CH/HC and OH/HO contacts. The UV-Vis spectral analysis of the SBF molecule
exhibit the absorption band in red shift and exposed the incidence of maximum transition
from nπ* excitation. The energy of the molecular orbitals was helpful to predict the
global reactivity descriptors, which represent that the synthesized molecules are
chemically hard substances with better kinetic stability and electron contributing

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capability. The molecular docking study of the SBF molecule has inhibitor activity against
the lung cancer protein as evident from the binding energy of –4.45 and –4.99 kcal/mol for
2ITU and 2ITO. This result identifies the inhibitory activity against lung cancer as
compared from experimental cytotoxic activity on A549 cell line.
Crystallographic data for the structure reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre as supplementary publication No.
CCDC-1871640. Copies of available material can be obtained free of charge via
www.ccdc.cam.ac.uk.
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